The invention concerns the method of manufacturing an intravenously tolerable immunoglobulin-G preparation.
Immunoglobulin-G preparations obtained from human plasma have been employed for many years to treat inherited and acquired immune-deficiency diseases. The immunoglobulin was initially obtained from human blood plasma by fractional precipitation with ethanol. These preparations, however, could be employed only intramuscularly since intravenous administration was accompanied by extremely serious side effects.
Dosage is limited in intramuscular administration. To obtain a high enough level of immunoglobulins for effective treatment it would accordingly be desirable to be able to make intravenously tolerable immunoglobulin-G preparations.
Various approaches to the problem have already been described. The immunoglobulin-G molecule has been split with enzymes or modified with chemical reagents for example. Other methods of obtaining intravenously tolerable preparations involve precipitation, adsorption and chromatography.
Reviews of the methods currently employed are provided in Krankenhauspharmazie 6, 5 (1985), 226-231 and in Der Bayerische Internist 9, 1 (1989), 36-44.
Although all the preparations described therein can be administered intravenously, they exhibit various drawbacks, depending on how they are prepared. The enzymatically split immunoglobulins for example have a severely curtailed intravascular half life and are incapable of triggering the complement activation requisite for effective defense against infections. Nor do the chemically modified preparations carry out the full range of biological functions due to their short intravascular half life and unsatisfactory complement activation.
To eliminate the aforesaid drawbacks, therefore, improved methods of manufacturing an intravenously tolerable unmodified immunoglobulin-G have been developed.
German A 3 039 855 for example discloses filtering over a polysulfone membrane, especially to remove polymeric substances (aggregates).
German A 3 641 115 proposes purification with polyethylene glycol to remove aggregates.
Neither of these processes, however, allows the elimination of proteolytic activities or vasoactive substances. These substances, which may occur in immunoglobulin-G preparations, include in particular prekallikrein activator, prekallikrein, kininogen and kallikrein. They are notoriously capable of causing serious side effects and especially of triggering hypotensive reactions (cf. M. F. Makula et al., Developm. biol. Standard 67 (1987), 257-65 and W. K. Bleeker et al, Vox Sang. 52 (1987), 281-290).
Attempts to remove proteolytic enzymes by adsorption on bentonite, silica gel, barium sultate, active carbon, or affinity exchangers have not succeeded in eliminating all the deleterious proteases and, depending on the method of manufacturing the immunoglobulin-G preparation, immunological and enzymatic tests have shown it still to contain varying levels of prekallikrein, kininogen and kallikrein even when the presence of any significant level of prekallikrein activator was impossible to identify.
The administration of such preparations, containing the aforesaid substances, to immunosuppressed patients has led to severe side effects even though the substances are highly tolerable intravenously in other applications. Thus, kallikrein can be released in the presence of prekallikrein even when no prekallikrein activator is detected and can release in turn the vasoactive bradykinin from the high molecular-weight kininogen. The result is a drop in blood pressure that is more or less marked in accordance with concentration. This must be taken into consideration in particular with patients with a malfunction in their inhibitor potential, as in the case of immunosuppression.
A generally applicable intravenously tolerable immunoglobulin-G preparation must accordingly be
a) free of aggregates, with an accordingly strictly low and unspecific complement-system activation, PA1 b) free of proteolytic enzymes, PA1 c) free of such vasoactive substances from the clotting and kinin systems as prekallikrein activator, prekallikrein, kininogen, kallikrein, Factor XI and Factor XII, and PA1 d) biologically intact, meaning that the structure of the Fc component of the immunoglobulin G is unaltered by the manufacturing process and that no chemical modification is employed.
The object of the present invention is to manufacture an intravenously tolerable immunoglobulin-G preparation that can be employed in all types of patients and is free of aggregates, vasoactive substances, and proteolytic activity from a starting material that contains an immunoglobulin. Examples of such a starting material are plasma with the coagulation factors removed or a serum fraction (Cohn's paste II or II-III) that contains immunoglobulin-G for example.
This object is attained in accordance with the invention by treating the starting material with 0.4-1.5% by volume of octanoic acid and subjecting it to chromatography.
The uses of octanoic acid are described in the literature (Biochemistry and Biophysics 134[1969], 279-84). Concentrations of approximately 6.8% by volume are employed to precipitate proteins from plasma or serum, leaving immunoglobulin-G in the supernatant.
It has surprisingly now been discovered that concentrations of 0.4 to I.5% by volume and preferably of 0.8 to 1.0% by volume of octanoic acid can be employed to eliminate specifically proteolytic enzymes and such compounds as prekallikrein, kallikrein and kininogen that trigger vasoactive reactions, from previously purified immunoglobulin-G solutions with no significant precipitation of protein.
The octanoic acid can then be centrifuged out of such a purified immunoglobulin-G solution or preferably filtered out while having calcium ions added to it.
The effectiveness of the octanoic acid treatment in removing proteolytic enzymes and vasoactive substances from immunoglobulin-G solutions (with a raw immunoglobulin-G fraction obtained from a Cohn procedure as described in Example 1 as a starting material) will be evident from Tables 1 and 2, wherein
++means strongly positive, PA0 I means positive, and PA0 -means negative.
TABLE 1 ______________________________________ Octanoic acid Proteolytic activity (% by volume) (units per liter) ______________________________________ 0.0 850 0.4 23 0.6 1 0.8 0 1.0 0 ______________________________________
TABLE 2 ______________________________________ Octanoic acid (% by vol) Prekallikrein* Kallikrein* ______________________________________ 0.0 ++ ++ 0.8 - - 1.0 - - 1.5 - - ______________________________________ *Determined by Ouchterlony's immunodiffusion. Starting from a raw immunoglobulinG fraction obtained by chromatography as described in Example 5.
As these results demonstrate, vasoactive substances and proteolytic enzymes can be removed from solutions that contain immunoglobulin-G.
Aggregated immunoglobulin-G molecules can be chromatographed out of the solution in a subsequent step.
Chromatography phases based on silica gel or a polymer are appropriate, and either cationic or anionic exchangers can be employed.
Preferred are chromatography phases that are appropriate for high-performance liquid chromatography (HPLC), a method that can be employed to economically and rapidly manufacture large volumes of immunoglobulin-G.
When such cation exchangers as CM-Accell(.RTM.), SP-Spherodex(.RTM.), SP-Trisacryl-LS(.RTM.) or Fraktogel-TSK-SP650(.RTM.) are employed, the salt concentration of the eluents is adjusted to 50-200 moles of sodium chloride and the pH to 4.0-6.0. In these conditions the aggregates will attach to the phase and the purified immunoglobulin-G will pass through the matrix without adhering to it.
The same method an also be employed with such anion exchangers as QMA-Accell(.RTM.) and DEAE-Spherosil(.RTM.) to remove aggregates from immunoglobulin-G solutions if the ion concentrationis adjusted to 0 to 50 mmoles of sodium chloride and the pH to 6.0 to 8.0.
It has also been demonstrated that the aforesaid chromatography phases can be employed to remove vasoactive substances, especially when cation exchangers or hydrophobic phases are employed Preferred for this purpose are chromatography phases based, as are CM-Accell(.RTM.), SP-Spherodex-M(.RTM.), and Polypropyl A(.RTM.), on silica gel or phase based, as are SP-Trisacryl-LS(.RTM.), on synthetic polymers
If a cation exchanger is employed, the ionic strength and pH of the eluent must allow the purified immunoglobulin-G fraction to travel through the column unimpeded while retaining the contaminants in the chromatography phase.
If CM-Accell(.RTM.) is employed, the buffer will for example have a concentration of 50-200 mmoles of sodium chloride and a pH of 4.0-6.0.
If the immunoglobulin is purified by hydrophobic interaction chromatography on Polypropyl A(.RTM.), the immunoglobulin-G solution will be attached to the phase with a high molar ammonium sulfate buffer, the contaminants will be rinsed out, and the immunoglobulin-G fraction will be eluted with a linearly decreasing ionic-strength gradient.
Table 3 illustrates the effectiveness of chromatography for removing vasoactive substances from solutions that contain immunoglobulin G.
TABLE 3 ______________________________________ IgG Fraction Prekallikrein* Kallikrein* ______________________________________ Untreated** ++ ++ SP chromatography - - CM chromatography - - Polypropyl A .RTM. neg neg chromatography - - ______________________________________ *Determined by Ouchterlony's immunodiffusion **Starting with a raw immunoglobulinG fraction obtained by chromatography as described in Example 5.
High-purity intravenously tolerable immunoglobulin-G preparations can accordingly be manufactured by treating them in accordance with the invention with octanoic acid and/or chromatography. The starting material can be a gamma-globulin fraction (Cohn's paste II or II-III or the corresponding solutions) obtained by conventional Cohn's fractionation as occurs in large quantities in the industrial manufacture of human albumin. The method in accordance with the invention can also be applied to chromatographically isolated immunoglobulin-G fractions and to those obtained by precipitation with ammonium sulfate or polyethylene glycol. These starting materials can be obtained from normal-donor pools and preferably from donors specially selected for high antibody titers against viral, bacterial or cellular antigens.
The starting material for the method in accordance with the invention is a raw fraction containing immunoglobulin G that is obtained by chromatography or by Cohn processes and is sterilized. Chromatography fractions usually contain approximately 0.5 to 1.5% of aggregates, which are preferably completely removed on such cation exchangers as CM-Accell(.RTM.) If the procedure starts with a Cohn's paste II or II-III that contains up to 5% of aggregates, they can be removed in accordance with the invention with CM-Accell(.RTM.) or QMA-Accell(.RTM.). Vasoactive substances can also be removed by chromatography as previously described herein.
If proteolytic enzymes are present, they can be removed initially with octanoic acid, although this is unnecessary for immunoglobulin-G solutions distinguished by high purity.
Since the plasma or serum fractions employed in the method in accordance with the invention are potentially contaminated with such human-pathogenic viruses as hepatitis A, B, non-A, non-B and human-immunodeficiency viruses, it is preferable to sterilize the immunoglobulin-G solutions. This can be done, preferably prior to chromatography, with .beta.-propiolactone, TNBP+Tween, TNBP+sodium cholate, or TNBP, optionally in combination with ultraviolet radiation. Especially preferred is sterilization with 0.03-0.1% of .beta.-propiolactone along with ultraviolet light.
The products manufactured in this way in accordance with the invention can be stored liquid or lyophilized.
The advantage of the method in accordance with the invention is that an extremely wide range of starting materials that contain immunoglobulin G can be simply and rapidly purified to the extent that the resulting pure products can be intravenously administered without side effects to all types of patients and especially to immunosuppressed patients.
Animal tests of the tolerability of the preparations manufactured in accordance with the invention in comparison with that of products manufactured according to the state of the art will now be described.
One model is the rat (cf. Bleeker et al., Vox Sang. 52[1987], 281-290), and the effects on its blood pressure and heart rate of vasoactive substances of the type that may be present in conventional products were studied.
Table 4 summarizes the results.
TABLE 4 ______________________________________ % Devi- Vasoactive substance Prepa- ation in Heart Prekalli- Kalli- Kinin- ration b.p. rate krein.sup.1) krein.sup.1) ogen.sup.1) ______________________________________ 1.sup.2) (ref.) -30 +6 + + + 2.sup.2) (ref.) -42 -8 ++ + + 3.sup.2) (ref.) -32 +2 ++ + + 4* (inv.) -2 -4 - - - 5* (inv.) -3 +3 - - - 6** (inv.) -4 +1 - - - 7** (inv.) 0 +1 - - - El. from 7*** -13 +10 ++ + + ______________________________________ .sup.1) Determined by Ouchterlony's immunodiffusion .sup.2) Raw immunoglobulinG fraction obtained by chromatography as described in Example 5. *Manufactured as described in Example 8. **Manufactured as described in Example 9. ***Column eluate from preparation 7 employed as a positive control (containing vasoactive substances).
The solution is considered tolerable at bloodpressure drops to -10%.
As the results of this test indicate, preparations purified over an ion exchanger in accordance with the invention occasion almost no deviations in blood pressure or heart rate, which documents their high tolerability in comparison with conventional preparations.
In another test conducted on dogs (by the method of M. F. Makula et al., Developm. biol. Standard 67 (1987), 257-65), the tolerablity of immunoglobulin-G solutions manufactured in accordance with the invention was tested against that of conventionally manufactured preparations. This model is mainly concerned with determining the effects of polymeric materials (aggregates) by measuring changes in cardiac output.
Table 5 summarizes the results.
TABLE 5 ______________________________________ Vasoactive % Devi- substance.sup.1) ation in Aggre- Pre- Prepa- Cardiac gates, kalli- Kalkinin- ration output % HPSEC krein krein ogen ______________________________________ 8.sup.2) (ref.) -50 1.1 + + + 9.sup.3) (ref.) -49 1.91 ++ + + 10* (inv.) 0 0.00 - - - 11** (inv.) -13 0.00 - - - 12*** (inv.) 0 0.00 - - - ______________________________________ .sup.1) Determined by Ouchterlony's immunodiffusion .sup.2) Raw immunoglobulinG fraction obtained by chromatography as described in Example 5. .sup.3) Raw immunoglobulinG fraction obtained by Cohn's procedure as described in Example 1. *Manufactured as described in Example 1 of the present invention. **Manufactured as described in Example 8 of the present invention. ***Manufactured as described in Example 5 of the present invention.
The solution is considered tolerable at cardiac output drops to -15%.
As this test also demonstrates, the preparations manufactured in accordance with the invention are higher in purity and accordingly freer of side effects than are the previously known products. The complete removal of aggregates, proteolytic substances and vasoactive compounds allows the manufacture of immunoglobulin-G preparations that can even be employed with immunosuppressed patients.